Architecture and control of exocytosis and endocytosis in excitable cells

National Heart, Lung, And Blood Institute

Investigators

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Abstract

Dozens of proteins control endocytosis and exocytosis in mammalian cells. The identity and roles of many of these proteins have been assigned through a combination of genetics, biochemistry, and electrophysiology. However, the spatial organization, heterogeneity, regulation, dynamics, and mechanisms of these proteins have not yet been determined. These data are key to understanding how proteins regulate membrane trafficking in healthy cells and malfunction in diseases. Thus, we aimed to map key proteins that act during endocytosis and exocytosis. To accomplish this, we developed a combination of high-throughput live cell imaging, super-resolution fluorescence imaging, and electron microscopy to directly visualize organelles. The primary mechanism of uptake in mammalian cells is clathrin-mediated endocytosis. To determine the nanoscale structure of clathrin-coated endocytic sites in living cells, we used a super-resolution correlative light and electron microscopy imaging (CLEM) method. This allowed us to image the nanometer-scale location of proteins in the context of their local cellular environment. Specifically, we imaged the plasma membrane of cells with super-resolution localization microscopy and transmission electron microscopy (TEM) of platinum replicas. From this work, we discovered that endocytic proteins distribute into distinct spatial zones (rings) in relation to the edge of the clathrin lattice. The presence or concentration of specific proteins within these rings changes at distinct stages of organelle development. We propose that endocytosis is driven by the recruitment, reorganization, and loss of proteins within these partitioned nanoscale zones. In an effort to understand the assembly and curvature of single clathrin-coated endocytic structures, we studied the specific geometric transitions in the clathrin coat with high-resolution 3D and cryogenic electron tomography. To accomplish this, platinum-replica EM was used to track and examine in detail how the lattices assemble and dynamically rearrange across eight different common cell lines. These studies showed that the clathrin lattice is capable of assembling first as a flat lattice that then can spontaneously curve into a dome without added energy or factors. These structural transitions drive coat assembly to reshape a transport vesicle and control cellular signaling pathways. Cryogenic electron tomography showed that flat lattices are disordered and contain many pentagons. We propose that the ordering of this lattice drives spontaneous curvature. Furthermore, we propose that flat lattices are held flat with adhesion forces to the extracellular matrix. The release of these forces and the physical properties of the membrane are needed to initiate curvature. This is a new updated mechanistic model of clathrin lattice curvature. To further improve our cryoET pipeline to image the plasma membrane, we refined the unroofing pipeline of cells grown on grids. By developing and building a new pressure-driven unroofing machine, we can now control and standardize plasma membrane unroofing protocols needed for this procedure across many cells and conditions. We additionally developed new methods to lift the top plasma membrane from cells onto grids to target the apical cellular surface. Finally, we tested and deployed chemically-activated fluorescent ferritin particles as new protein-based tags for marking proteins in cryoET images. This pipeline provides a system for specifically marking and imaging the structure of single proteins at the plasma membrane of mammalian cells. Conformational changes in CLC have been shown to regulate triskelion assembly in solution, yet the nature of these structural changes and their effects on lattice growth, curvature, and endocytosis in cells are unclear. Here, we developed a correlative fluorescence resonance energy transfer (FRET) and platinum replica electron microscopy method, named FRET-CLEM. With FRET-CLEM, we measured conformational changes in proteins at thousands of individual morphologically distinct clathrin-coated structures across cell membranes. We found that the N-terminus of CLC moves away from the plasma membrane and triskelion vertex as lattices curve. Preventing this conformational switch with acute chemical tools inside cells increased clathrin structure sizes and inhibited endocytosis. Therefore, a specific conformational switch in CLC regulates lattice curvature and endocytosis in mammalian cells. One of the major signaling pathways in human cells needed for growth, differentiation, and homeostasis is the epidermal growth factor receptor (EGFR) pathway. Here, extracellular growth factor ligands activate plasma membrane receptor kinases to generate intracellular signals that modulate gene expression programs. EGFR is a common target for cancer therapy. The amount of active EGFR on the cell surface is thought to be controlled by the uptake of the receptor by clathrin-mediated endocytosis. We discovered that activation of the EGFR receptor causes a massive increase in the number and size of flat clathrin lattices in the plasma membrane. These flat clathrin lattices cluster the EGFR receptor and contain the Beta5 integrin. Manipulation of EGFR, Src kinase, and the integrin by drugs or knockdown prevented the formation of flat clathrin lattices needed for signaling of the receptor. Thus, we propose that flat clathrin lattices act as signaling hubs to co-cluster and control these three interconnected signaling systems at the plasma membrane of human cells. To further examine this system, we screened proteins that associate with clathrin lattices before and after growth factor stimulation. We found that specific receptors, including FGFR1 and LDLR, are co-recruited into clathrin sites with EGF. Other receptors, including G-protein coupled receptors, were not recruited. A panel of important endocytic proteins are also recruited, including Dyn2, EPS15, Grb2, and Cbl. These data indicate that clathrin acts as a more global signaling hub to collect and activate related and unrelated receptors at the plasma membrane of human cells. Furthermore, these parallel pathways offer new therapeutic treatments for cancers driven by mutations or overexpression of EGFR. Another endocytic and signaling organelle in human cells is caveolae. Caveolae are 80-nm diameter coated vesicles that invaginate into the cytosol from the plasma membrane. Their diverse functions span from endocytosis to signaling, regulating key cellular processes including lipid uptake, pathogen entry, and membrane tension. Caveolae undergo shape changes from flat to curved. We developed a correlative multi-color stimulated emission depletion (STED) fluorescence and platinum replica EM imaging (CLEM) method to image caveolae-associated proteins at caveolae of different shapes at the nanoscale. Caveolins and cavins were found at all caveolae, independent of their curvature. EHD2, a classic caveolar neck protein, was strongly detected at both curved and flat caveolae. Both pacsin2 and the regulator EHBP1 were found only at a subset of caveolae. Pacsin2 was localized primarily to areas surrounding flat caveolae, whereas EHBP1 was mostly detected at spheres. Contrary to classic models, dynamin was absent from caveolae and localized only to clathrin-coated structures. Cells lacking dynamin showed no substantial changes to caveolae, suggesting that dynamin is not directly involved in caveolae curvature. Together, we provide a mechanistic map for the molecular control of caveolae shape by eight of the major caveolae-associated coat and regulatory proteins. We propose a model where caveolins, cavins, and EHD2 assemble as a cohesive structural unit regulated by more intermittent associations with pacsin2 and EHBP1. These complexes can flatten and curve, capturing membrane to enable lipid and protein traffic and changes to the surface area of the cell. Many chronic diseases, including cancer, heart disease, and lung disease, are driven by the mis-trafficking of plasma membrane receptors. Many approved drugs in humans specifically target these receptors. Understanding how receptors interact with trafficking vesicles that transport them to and from the membrane is key. These experimental studies aim to reveal the structure and dynamics of exocytic and endocytic vesicles in health and disease at the cellular and molecular levels. By doing so, these studies address the root causes of chronic diseases affecting the heart, lung, blood, endocrine, and nervous systems, and seek to identify new avenues for treatments of these disorders.